The BvgAS signal transduction system coordinately regulates genes and operons involved in the Bordetella infectious cycle. Recent results indicate that the Bordetella virulence regulation is more complex, and interesting, than we had previously imagined. As opposed to mediating a biphasic transition, BvgAS appears to control a spectrum of gene expression states. We have also discovered the BvgAS regulates type III secretion, which functions at least in part to modulate host immunity. In the course of studying Bvg AS, type III secretion, and other virulence associated factors and phenotypes, we have developed genetic methods for manipulating Bordetella genomes and a variety of animal models for studying infection and the infectious cycles. The experiments outlined in this application are a direct extension of our most interesting observations to date. They represent a desire to understand the biology of the bordetellae from the perspectives of virulence gene regulation and the evolution of bacterial pathogenesis. In Aim 1 we will conduct a genetic and phylogenetic analysis of signal responsiveness by the BvgAS virulence control system. Bvg-regulated loci display markedly diverse patterns of gene expression which we hypothesize reflects a requirement for differential gene expression throughout the infectious cycle. We will characterize relationships between temporally and spatially controlled transcription levels of Bvg-regulated genes, determine if key characteristics of Bvg-mediated signaling are phylogenetically conserved, and identify sequences that determine signal responsiveness. In Aim#2, transcription of Bvg-regulated genes will be measured in vivo and requirements for differential gene expression during the infectious cycle will be examined. We will test the hypothesis that differential gene expression at different sites in the respiratory tract results in adaptation to specific niches and facilitates both infection and transmission. Aim #3 will focus on the regulation, phylogenetic conservation and comparative function of type III secretion by Bordetella subspecies. In Aim 4, we will use high density DNA microarrays to identify the entire complement of Bvg-regulated genes and investigate their functions in animal models of infection and transmission. We will test the hypothesis that the Bvg regulon is multiphasic by conducting a genome-wide expression analysis. Differences in expression profiles between B. pertussis, B. parapertussis and B. bronchiseptica will also be determined in an effort to understand subspecies-specific differences in Bordetella-host interactions.